Nutrafoods (2016) 15:111-121 Original Research DOI 10.17470/NF-016-1030-2 Process optimization for the preparation of apple wine with analysis of its sensory and physico-chemical characteristics and antimicrobial activity against food-borne pathogens

Vikas Kumar1, Vinod K. Joshi2, Gitanjali Vyas3, N.S. Thakur2, Nivedita Sharma3 Keywords: Apple, Tea, Apple tea wine, Correspondence to: Saccharomyces cerevisiae, Vikas Kumar Natural fermentation, [email protected] Functional properties

Abstract Apple tea wine fermented with S. cerevisiae var. el- lipsoideus had higher sensory scores for most at- Method for preparing apple tea wine using differ- tributes than naturally fermented apple tea wine. ent types of tea at different concentrations were Different treatments were clustered based on tea optimized and the physico-chemical, antimicrobial concentration as a function of fermentation with and sensory characteristics of the wine analysed. All S. cerevisiae var. ellipsoideus unlike the natural fer- characteristics were found to be directly propor- mentation where no clear clustering trend was tional to the concentration of tea fermented natu- observed. The type of fermentation influenced rally or with Saccharomyces cerevisiae var. ellipsoi- the quality of the wine as separate clusters for deus. In both types of fermentation, CTC (crush, the different fermentations were observed during tear and curl) tea-based apple tea wine received combined cluster analysis of the different types of significantly higher quality scores (p≤0.05). Bet- wine. Our results demonstrated that the best apple ter results in terms of ethanol, higher alcohol con- tea wine was made with 4 g CTC tea/100 ml apple centrations and antimicrobial activity were found juice and fermented with S. cerevisiae var. ellipsoi- with 4 g tea/100 ml apple juice than with other deus, and showed potential as a functional product concentrations, particularly 5 g tea/100 ml apple which also demonstrates the medicinal properties juice. All apple tea wines showed antimicrobial ac- of tea. tivity (inhibition zone >7 mm) against Escherichia coli (IGMC), Enterococcus faecalis (MTCC 2729), Introduction Listeria monocytogenes (MTCC 839), Staphylococcus aureus (MRSA 252) and Bacillus cereus (CRI). As economic status has improved, growing interest in and well-being has resulted in increased intake of natural foods. Tea, a non-alcoholic bev- 1Assistant Professor, Food Technology and Nutrition, School of Agriculture, Lovely Professional University, Phagwara, Punjab erage consumed worldwide and prepared from tea 144411, India leaves ( L.) is gaining popularity phone: +919418653296 as an important ‘health drink’ and is consumed 2Department of Food Science and Technology, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal by up to two thirds of the world’s population. Pradesh 173230, India. Among the 700 chemical constituents present in 3Department of Basic Sciences, Dr. Y.S. Parmar University of Hor- ticulture and Forestry, Nauni, Solan, Himachal Pradesh 173230, tea leaves, flavonoids, amino acids, vitamins (C, India. E, K), and polysaccharides are considered

Received: September 13, 2015 / Accepted: December 28, 2015

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important for human health. The caffeine (3% by crobial activities of have been less weight) in tea provides a stimulating effect [1]. studied than its beneficial health properties. These Polyphenols in tea are the most important con- antimicrobial activities develop during the natu- stituents and act as antioxidants, known to play ral microbial fermentation process with tea leaves a significant role in human health. is as substrates. The antimicrobial components pro- considered to be a good fermentation medium be- duced during the fermentation process have shown cause its contains proteins, amino acids, inhibitory effects against several food-borne and volatile compounds, lipids, enzymes and, more pathogenic bacteria as reported by Sreeramulu et importantly, polyphenols [2]. al. [22]. Its acidity and low alcohol content allow Wine consumed in moderation is considered a to resist contamination by most air- safe and healthy drink and is an important adjunct borne moulds and bacterial spores. The beverage is to the diet [3]. Different studies have shown the brewed at home so preparation conditions are gen- beneficial effects of wine consumption due to the erally not sterile. However, most tests indicated a presence of phenolics and alcohol, which protect low rate of contamination from spoilage and path- the human body from attack by free radicals and ogenic , suggesting that kombucha increase HDL levels, respectively [4, 5]. Phenolic has antimicrobial properties against pathogenic compounds also play a major role in enology as and other harmful microorganisms [23]. Some they are responsible for the colour and flavour of studies have reported that the antimicrobial activ- red and white wine. In addition to their bacteri- ity of kombucha made with a low concentration cidal role, phenolics are responsible for the ‘French of tea (4.4 g/l) was attributable to the acetic acid paradox’ whereby antioxidants and vitamins are content [10]. Indeed, the systematic investigation apparently protective against cardiovascular dis- of Sreeramulu et al. [22] demonstrated that the ease [6]. antimicrobial activity of kombucha was not due to Apple juice has been fermented in Eastern Mediter- organic acids, ethanol, proteins or tannins present ranean areas for more than 2000 years to obtain a in tea or their derivates. pleasant alcoholic beverage [7]. It is also fermented The chemical constituents of tea and the phyto- to produce cider, a sparkling and refreshing fruit- chemical potential of apple juice can both be used flavoured drink consumed in many countries [5, to improve the quality of wine. Siby and Joshi 8], as well as wine and brandy. Kombucha or tea [24] supplemented apple wine with spice extracts cider (a traditional fermented tea product) is often which increased the polyphenolic content and also drunk for medicinal purposes due to the acetic acid enhanced antimicrobial activity. In light of the formed during fermentation and has been used in health benefits of tea, the importance of apples in Russia for several centuries [9]. Alcohol is also pro- the wine industry and the role of different micro- duced during fermentation of kombucha [10]. flora in wine fermentation, the aim of this study The increasing antimicrobial resistance of patho- was to improve the physico-chemical, antimicro- gens isolated from humans and animals, com- bial and sensory properties of apple tea wine pro- bined with the increasing consumer awareness of duced by fermentation with Saccharomyces cerevi- chemical food preservatives, necessitates research siae var. ellipsoideus and by natural fermentation. for more efficient antimicrobials with fewer side- The results of our study are presented below. effects on human health. Different plant extracts were examined for antimicrobial activity against Materials and methods pathogens by Papadopoulou et al. [11]. Tea ex- tracts have exhibited antioxidant [12], antimuta- Materials genic [13, 14], anticarcinogenic [15], antibacterial Apples (Golden variety) were procured from the [16], antiviral [17], antifungal [18] and antitumor local market in Solan (Himachal Pradesh, India). activity [19, 20]. Natural antibacterial agents have Orthodox tea (Dhauladhar, natural organic or- been increasingly applied for the biological pres- thodox ) was procured from HPKV, ervation of food in recent years [21]. The antimi- Palampur (Himachal Pradesh, India),

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(Himalayan brew, Kangra special herbal tea) was wines were analysed for different physico-chemical procured from the local market in Palampur, and properties: TSS, titratable acidity (as malic acid), CTC tea (Tajmahal) was procured from the local pH, according to standard methods [25], market in Solan. The yeast strain S. cerevisiae var. and ethanol measured colourimetrically by the po- ellipsoideus (UCD 595) was procured from the In- tassium dichromate method [26]. Volatile acidity dian Institute of Horticulture Research, Bangalore was estimated by the titration methods described (Karnataka, India). was procured from the by Amerine et al. [27]. The content of higher alco- local market. Pectin esterase was procured from hols was estimated using the method of Guymon M/S Triton Chemical, Mysore (India) and used at et al. [28]. a concentration of 0.5%. The antimicrobial activity of apple juice, apple tea wine and tea cider was determined against all Methods the test microorganisms, that is, Escherichia coli of tea leaves with apple juice were pre- (IGMC), Enterococcus faecalis (MTCC 2729), Lis- pared by boiling different concentrations of tea in teria monocytogenes (MTCC 839), Staphylococcus apple juice for 3 min at 100°C. The concentra- aureus (MRSA 252) and Bacillus cereus (CRI), by tions (2, 3, 4 and 5 g per 100 ml apple juice) and the well diffusion method [29] under aerobic con- types of tea (CTC, orthodox and herbal) varied, ditions. resulting in 12 different combinations. Infusions A semi-trained panel of judges analysed the sen- were filtered through sieves and used as fermen- sory characteristics of different apple tea wines us- tation media. To each infusion, 0.1% diammo- ing composite scoring and the hedonic rating test nium hydrogen phosphate (DAHP) as a nitrogen as described by Amerine et al. [27] and Joshi [30]. source and 0.5% pectinesterase for clarification were added, respectively. The total soluble solids Statistical analysis (TSS) value was raised to 20°B by adding sugar, Data on the physico-chemical and functional and sulphur dioxide (100 ppm) was added to kill properties of the apple tea wine were subjected wild microorganisms. After 2 h, each must was in- to analysis of variance using a completely rand- oculated with 5% second generation 24-hour-old omized design (CRD) and the means with critical S. cerevisiae var. ellipsoideus and left to ferment at differences reported. Cluster analyses of data us- room temperature. Combinations were fermented ing SPSS 16.0 software was carried out. The results three times at the same conditions so that the ef- were plotted as dendrograms. Statistical analysis of fect of multiple replicate fermentation processes data obtained from sensory evaluation of the wine on the quality of the wine could be studied. was carried out using a randomized block design Fermentation was considered complete when a (RBD) as described by Cockrane and Cox [31]. stable TSS had been reached. Air locks were fit- Results are reported in the tables. ted to the mouths of the glass bottles close to the end of fermentation. After fermentation was com- Results and discussion plete, the wines were racked, filtered and poured into 200 ml bottles with 2.5 cm head-space, fol- Fermentation with S. cerevisiae lowed by crown corking and mild pasteurization, var. ellipsoideus and used for the analysis of physico-chemical, Effect of different types of tea antimicrobial and sensory properties. Wines fer- Different types (orthodox, herbal and CTC) of mented naturally at room temperature using the tea were used in order to study their effect on the same methods but without inoculation were also quality of apple tea wine. The rate of fermentation produced and bottled. was higher in wine made with herbal tea (1.11) than the other tea types, possibly due to the lower Analysis total phenol content in herbal tea [32]. The high- Falls in TSS (°B) were monitored at appropriate est TSS (7.05°B), reducing sugar content (396 time points during fermentation. After bottling, mg/100 ml) and total sugar content (1.20%) were

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observed in wine made from CTC tea due to the alcoholic beverages led to a decrease in the Brix low fermentability of the musts because of their drop rate, although changes in fermentation effi- high total phenolic content. The highest titratable ciency were marginal. A significant (p≤0.05) in- acidity (0.75%) was observed in wine made with crease in the content of reducing sugars (from 275 CTC tea and the lowest in wine made from or- to 352 mg/100 ml) and total sugars (from 1.10% thodox tea (0.68%). Volatile acidity did not differ to 1.14%) (Table 2) was observed with an increase significantly (p≤0.05) among the wines prepared in tea concentration from 2 to 4 g/100 ml apple from different types of tea. However, significantly juice, which might be due to the inhibiting effect higher (p≤0.05) ethanol content (8.98%) was ob- of total phenols on alcoholic fermentation which served in wine made with herbal tea, which might increased as tea concentration increased, as report- be due to the high rate of fermentation and low ed earlier [32]. Amerine et al. [27] found that the total phenolic content of herbal tea extracts, both natural phenols present in grapes and wine includ- important in fermentation as described earlier. The ing tannins, total phenols and pigment polymers, lowest alcohol content was observed in wine made are inhibitory to yeast and bacteria. In addition, with orthodox tea and herbal tea (125 mg/l), while a non-significant effect of different concentrations the highest was found in wine made with CTC tea of tea was also observed on the TSS, titratable (163 mg/l) (Table 1), possibly due to the compo- acidity and volatile acidity of apple tea wine. sition of CTC tea which is reported to be rich in The difference in alcohol content among the dif- oxidized products such as and thearu- ferent concentrations of tea is likely related to dif- bigins [33], which might have contributed to the ferences in the fermentability of the must. Results formation of higher alcohols. Higher alcohols are revealed that alcohol content ranged from 8.36% synthesized during fermentation from oxo-acids to 8.82% (v/v) in wine prepared from the differ- that are derived as by-products from amino acid ent concentrations of tea, which demonstrates that and glucose metabolism [34]. The highest levels of fermentation was completed almost to dryness. total phenols, epicatechin, caffeine, protein con- Since table wine has an alcohol content of 7–14% tent and amino acids were observed in wine made [27], all wines prepared in the present study fall with CTC tea and the lowest were in wine made into this category. The content of higher alcohols with orthodox tea as reported earlier [32]. There increased with increasing tea concentration: wine was a non-significant difference in antioxidant made from 5 g tea/100 ml apple juice was found activity among the different tea-based apple tea to have 159 mg/l higher alcohols. Higher alcohols wines as reported earlier [32]. are synthesized during fermentation from oxo- acids that are derived as by-products from amino Effect of different concentrations of tea acid and glucose metabolism [34], as mentioned The concentration of each tea was varied from 2 to earlier. An increase in concentration of tea from 2 5 g/100 ml apple juice to determine the effect of to 5 g was accompanied by a significant (p≤0.05) concentration on the quality of the apple tea wine. increase in total phenols, epicatechin, proteins, An increase in tea concentration enhanced the rate amino acids and caffeine in apple tea wine, as re- of fermentation (Table 2), likely due to the high ported earlier [32]. A non-significant increase in content of amino acids (2–4% dry basis) which are antioxidant activity was observed with increased an essential nutrient for yeast growth, accelerating tea concentrations, as reported earlier [32]. fermentation. Nitrogen is used by the yeast for synthesizing structural proteins, enzymes, nucleic Natural fermentation acids and pyrimidine nucleotides [35]. Assimilable Effect of different types of tea nitrogen has been shown to increase sugar fermen- Different types of tea (orthodox, herbal and CTC) tation and ethanol formation [35, 36]. However, were used in order to study their effects on the the results of the present study are contrary to the physico-chemical and functional properties of findings of Jayasundara et al. [37] who reported apple tea wine. Wine made with herbal tea had the use of higher concentrations of tea to produce the highest rate of fermentation (1.20), while that

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Type of tea Rate of TSS (°B) Reducing Total sugars Titratable Volatile acidity Ethanol Higher alcohols fermentation sugars (%) acidity (% acetic acid) (% v/v) (mg/l) (°B) 24 h (mg/100 ml) (% malic acid) Orthodox 1.06 6.75 265 1.11 0.68 0.027 8.23 125 Herbal 1.11 6.75 360 1.06 0.69 0.023 8.98 125 CTC 0.99 7.05 396 1.20 0.75 0.029 8.36 163 CD (p>0.05) 0.05 NS 12 0.01 0.03 NS 0.26 2 CD critical difference,CTC crush, tear and curl, NS not significant, TSS total soluble solids Table 1 - Changes in the physico-chemical characteristics of apple tea wine fermented with Saccharomyces cerevisiae var. ellipsoi- deus as affected by different types of tea

Concentration of tea Rate of TSS (°B) Reducing Total Titratable Volatile acidity Ethanol Higher (per 100 ml apple juice) fermentation sugars sugars (%) acidity (% acetic acid) (% v/v) alcohols (°B) 24 h (mg/100 ml) (% malic acid) (mg/l) 2 g 0.98 6.60 275 1.10 0.71 0.026 8.44 112 3 g 1.06 6.87 324 1.13 0.69 0.025 8.47 136 4 g 1.07 7.00 352 1.14 0.71 0.027 8.82 142 5 g 1.10 6.93 344 1.13 0.70 0.028 8.36 159 CD (p>0.05) 0.06 NS 13 0.01 NS NS 0.26 2 CD critical difference,CTC crush, tear and curl, NS not significant, TSS total soluble solids Table 2 - Changes in the physico-chemical characteristics of apple tea wine fermented with Saccharomyces cerevisiae var. ellipsoi- deus as affected by different concentrations of tea

made with CTC tea had the lowest (0.79), pos- the two treatments. The highest ethanol content sibly due to the low polyphenolic content of the (8.31%) was observed in wine made with herbal CTC tea-based must, which had an inhibitory ef- tea and the lowest (7.63%) in wine made with or- fect on alcoholic fermentation [27]. The highest thodox tea (Table 3). The lowest content of higher TSS (7.80°B) and reducing sugar content (367 alcohols (191 mg/l) was observed in wine made mg/100 ml) were observed in wine made with with orthodox tea and the highest (304 mg/l) in CTC tea and the lowest in wine made with herbal wine made with CTC tea (Table 3), possibly due tea owing to the fermentability of these different to the composition of CTC tea, as discussed ear- tea-based musts, as discussed earlier. The lowest lier. The highest concentrations of total phenols, total sugar content (0.79%) was observed in wine epicatechin, protein content and amino acids were made with herbal tea and the highest (1.94%) in observed in wine made with CTC tea and the wine made with orthodox tea. There were non-sig- lowest in wine made with herbal tea, as reported nificant differences in titratable acidity among the earlier [32]. The highest caffeine was observed in different types of tea (Table 3). The highest volatile wine made with CTC tea and the lowest in wine acidity (0.041%) was observed in wine made with made with orthodox tea, which was possibly due CTC tea and the lowest (0.029%) in wine made to the contribution of tea leaves (w/w), as reported with herbal tea due to the rates of fermentation of earlier [32].

Type of tea Rate of TSS (°B) Reducing Total su- Titratable Volatile acidity Ethanol Higher fermentation sugars gars (%) acidity (% (% acetic acid) (% v/v) alcohols (°B) 24 h (mg/100 ml) malic acid) (mg/l)

Orthodox 1.11 7.30 237 1.94 0.76 0.034 7.63 191 Herbal 1.20 6.90 99 0.79 0.81 0.029 8.31 199 CTC 0.79 7.80 367 1.85 0.81 0.041 7.91 304 CD (p>0.05) 0.08 0.32 11 0.02 NS 0.007 0.19 2 CD critical difference,CTC crush, tear and curl; NS not significant, TSS total soluble solids

Table 3 - Changes in the physico-chemical characteristics of apple tea wine naturally fermented as affected by different types of tea

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Effect of different concentrations of tea Effect of type of fermentation The concentration of each tea was varied from 2 on the physico-chemical and functional to 5 g/100 ml apple juice to study the effect on properties of apple tea wine the physico-chemical and functional properties The rate of fermentation was lower in naturally fer- of apple tea wine. Must made with 5 g tea/100 mented apple tea wines compared to apple tea wine ml apple juice had a higher rate of fermentation fermented with S. cerevisiae var. ellipsoideus, possibly (1.15), closely followed by must made with 4 g due to mixed microbial fermentation during natural tea/100 ml apple juice (1.13), possibly due to the fermentation, where both yeast and bacteria could presence of a high quantity of amino acids which have played a role. In addition, secretion of a killer increased with increased tea concentration (Ta- factor by the wild yeast found in the early stage of ble 4), as discussed earlier. The data revealed that must fermentation could have inhibited the process the difference in TSS was non-significant among and thereby lowered the rate of fermentation as re- the different tea concentrations, while varying ported by Farris et al. [39]. Higher TSS and greater the concentration of tea from 2 to 4 g resulted content of reducing and total sugars were observed in a significant (p≤0.05) decrease in the content in naturally fermented wines compared to those of reducing sugars (from 293 to 177 mg/100 ml) fermented with S. cerevisiae var. ellipsoideus due to and total sugars (from 2.02% to 1.16%); how- differences in fermentability. The high acidity and ever, slightly higher values for both sugars were volatile acidity of naturally fermented wine (Tables observed for 5 g tea/100 ml apple juice (Table 4). 3 and 4) might be due to the presence of natural The highest titratable acidity (0.83%) and highest microflora including both yeast and bacteria. The volatile acidity (0.046%) were observed in wine higher volatile acidity of wine naturally fermented made with 5 g tea/100 ml apple juice as compared is due to the presence of mixed microflora other to the other concentrations studied. The highest than Saccharomyces as reported by Caridi et al. [40]. ethanol production was recorded in wine made These authors described the high volatile acidity of with 2 g tea/100 ml apple juice (8.19%) (Table 4), wine fermented with Hanseniaspora quilliermondii likely due to the low total polyphenolic content of whose acetogenic property made the yeast unsuit- the tea and the presence of natural yeast identified able for wine production as it may encourage the as S. cerevisiae, which might have boosted alco- growth of acetic acid bacteria during fermentation holic fermentation [38]. The data show that an (lesser growth increases acetic acid concentrations increase in tea concentration from 2 to 5 g/100 and consequently volatile acidity). ml apple juice resulted in an increased content of Equal amounts of ethanol were produced by both higher alcohols (from 190 to 277 mg/l) (Table 4). fermentation processes, possibly due to the domi- An increase in total phenols, epicatechin, protein nance of S. cerevisiae var. ellipsoideus over natural content, amino acids, caffeine content and an- microflora during the initial stages of natural fer- tioxidant activity of the wine was also observed mentation. Heard and Fleet [41] reported that S. with increased tea concentration, as reported ear- cerevisiae dominated wine fermentation but that lier [32]. there was significant growth of other yeast species

Concentration of tea Rate of TSS (°B) Reducing Total su- Titratable Volatile acidity Ethanol Higher (per 100 ml apple juice) fermentation sugars gars (%) acidity (% (% acetic acid) (% v/v) alcohols (°B) 24 h (mg/100 ml) malic acid) (mg/l)

2 g 0.84 7.33 293 2.02 0.77 0.032 8.19 190 3 g 0.99 7.27 263 1.69 0.77 0.031 8.09 212 4 g 1.13 7.27 177 1.16 0.80 0.031 7.61 246 5 g 1.15 7.47 205 1.24 0.83 0.046 7.91 277 CD (p>0.05) 0.09 NS 12 0.02 0.04 0.008 0.22 3 CD critical difference,NS not significant,TSS total soluble solids Table 4 - Changes in the physico-chemical characteristics of apple tea wine naturally fermented as affected by different concentra- tions of tea

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such as Kloeckera apiculata, Candida stellata, Can- ent components of apple tea wine (polyphenolics, dida colliculosa, Candida pulcherrima and Hansenu- alcohol and citric acid) produced using different la anomala. These species are susceptible to increas- concentrations of tea (Fig. 1) showed that an in- ing alcohol levels and are not as alcohol tolerant crease in tea concentration (from 2% to 5% with as S. cerevisiae [42]. There was a greater content of ) resulted in a slight increase in antimicrobial higher alcohols in the naturally fermented wine activity against all tested microorganisms, possi- compared to those fermented with S. cerevisiae var. bly due to an increased concentration of phenols. ellipsoideus due to the presence of mixed microflora Phenolic compounds may affect the growth and during natural fermentation. The highest values for metabolism of bacteria, and have an activating or total phenols, epicatechin, proteins, amino acids inhibiting effect on microbial growth depending and caffeine were recorded in naturally fermented on composition and concentration [44]. The in- wine and were higher than in wines fermented with hibitory effect of phenolic compounds could be S. cerevisiae var. ellipsoideus, as reported earlier [32]. due to their adsorption to cell membranes, inter- This may be due to the low fermentability of natu- action with enzymes, substrate and metal ion dep- rally fermented wines which might have resulted in rivation [44, 45]. It was also observed that none less degradation of caffeine than in wines ferment- of the concentrations of ethyl alcohol (5–50%) ed with S. cerevisiae var. ellipsoideus. Our results are tested exhibited antimicrobial activity against any in line with the findings of Malbasa et al. [43] who of the test microorganisms. Similar findings were reported that during kombucha fermentation, caf- also observed by Boban et al. [46] who reported feine content decreases continuously and is inde- that among the different components of wine, eth- pendent of tea concentration. anol had the lowest antimicrobial activity against all tested microbes (E. coli [ATCC 25922] and Antimicrobial activity Salmonella enterica [ATCC 13076]). An increase Effects of different concentrations of polyphenolics, in acid concentration (from 0.25% to 1.00%) was acid and ethyl alcohol on antimicrobial activity accompanied by an increase in antimicrobial activ- Analysis of the antimicrobial activity of the differ- ity against all tested microorganisms in the present

12 11 10 Bacillus cereus 9 8 Enterococcus faecalis 7 6 Escherichia coli 5 4 3 Listeria monocytogenes Inhibition zone (mm) Inhibition 2 1 Staphylococcus aureus 0

2% CTC tea3% CTC tea4% CTC tea5% CTC tea 5% ethanol 1% citric acid 10% ethanol15% ethanol20% ethanol 0.25% citric0.5% acid citric0.75% acid citric acid

Figure 1 - Effect of the different components (concentration of tea [for polyphenolics], citric acid [acidity] and alcohol) of apple tea wine on antimicrobial activity. CTC crush, tear and curl

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Test CTC tea (per 100 ml apple juice) Herbal tea (per 100 ml apple Orthodox tea (per 100 ml apple juice) juice)

2 g 3 g 4 g 5 g 2 g 3 g 4 g 5 g 2 g 3 g 4 g 5 g Apple tea wine fermented with Saccharomyces cerevisiae var. ellipsoideus Escherichia coli 8.50 9.25 10.75 9.00 9.00 9.25 10.00 11.25 8.00 8.50 10.00 10.50 Staphylococcus aureus 7.00 7.75 8.00 8.00 8.00 8.50 8.75 9.00 6.50 7.50 7.00 7.50 Bacillus subtilis 9.00 9.00 9.50 9.50 9.00 9.00 9.50 9.50 7.00 8.50 9.00 6.00 Bacillus cereus 9.00 9.50 10.00 10.00 7.00 8.50 9.00 9.25 7.00 7.50 8.00 9.00 Enterococcus faecalis 7.00 8.00 8.50 9.00 7.00 7.50 7.50 8.50 6.00 6.50 6.75 7.00 Natural fermentation Escherichia coli 8.50 8.50 9.50 10.00 8.25 9.00 9.50 9.50 7.25 7.75 9.25 9.50 Staphylococcus aureus 7.50 8.00 9.50 8.00 7.00 7.50 7.50 8.50 6.50 6.50 7.00 8.00 Bacillus subtilis 7.00 9.50 10.00 9.00 7.00 7.50 8.50 9.00 9.50 8.50 9.50 10.00 Bacillus cereus 8.50 9.00 9.00 9.50 7.00 8.00 9.50 9.50 8.50 8.00 10.50 11.00 Enterococcus faecalis 7.00 7.00 8.00 8.00 6.00 7.00 8.00 8.50 7.50 7.50 8.00 9.50 CTC crush, tear and curl Table 5 - Effect of different concentrations and types of tea on antimicrobial activity (inhibition zone in mm) of apple tea wine fermented with Saccharomyces cerevisiae var. ellipsoideus and by natural fermentation

study. The results of the present study are in line duced with different types of tea, concentrations with the findings of Waite and Daeschel [47] who and types of fermentation (Table 5). However, examined the antimicrobial activity of four wine all wines exhibited antimicrobial activity (inhibi- parameters (pH, titrable acidity, sulfur dioxide tion zone >7 mm) against all pathogenic microbes and ethanol) in various combinations against E. tested. This might be due to the presence in the coli O157:H7 and S. aureus, and reported that pH wines of organic acid and total phenols which are was the most critical factor in predicting inactiva- both important in antimicrobial activity, as shown tion of tested pathogens. experimentally (Fig. 1).

Antimicrobial activity of apple tea wine fermented Sensory analysis naturally and with S. cerevisiae var. ellipsoideus The sensorial composite scoring of apple tea wine No clear-cut trend was observed in the antimi- fermented with S. cerevisiae var. ellipsoideus and by crobial activity of the various apple tea wines pro- natural fermentation is compared in Fig. 2.

Appearance Overall impression Colour

Astringency Aroma and bouquet Fermentation with Saccharomyces cerevisiae var. ellipsoideus

Natural fermentation Bitterness Volatile acidity

Flavour Total acidity

Body Sweetness

Figure 2 - Sensory evaluation of apple tea wine fermented by different types of fermentation

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Fermentation with S. cerevisiae var. ellipsoideus wines made with 4 or 5 g tea/100 ml apple juice. showed higher scores for most sensory attributes However, cluster analysis failed to separate type of than naturally fermented wine except for volatile tea into a cluster, indicating that type of tea did acidity, total acidity, sweetness and astringency. not influence the physico-chemical and functional Scores showed that wines prepared with S. cerevi- properties of the wine. However, in case of wine siae var. ellipsoideus and by natural fermentation fermented naturally, type of tea or tea concentra- fell into the ‘standard’ category [27]. tions did not affect the physico-chemical or func- tional properties. Nevertheless, 5 g tea is an out- Cluster analysis lier as shown in Fig. 3B. The distinctness of this A dendrogram of the different treatments of apple treatment is also shown by the differences among tea wine according to the physico-chemical and the physico-chemical and functional properties functional properties of wine fermented with S. of the treatment. Cluster analysis successfully cerevisiae var. ellipsoideus is shown in Fig. 3A. grouped the two different types of fermentation Cluster analysis grouped wine made with 2 and into separate clusters as shown in Fig. 3C. One 3 g tea/100 ml apple juice into one cluster and cluster comprises wine fermented with S. cerevisiae wine made with 4 and 5 g tea/100 ml apple juice var. ellipsoideus, while another comprises naturally into another cluster, indicating that the physico- fermented wine. Cluster analysis further revealed chemical characteristics of wines made with 2 or 3 that the type of fermentation influenced the phys- g tea/100 ml apple juice are similar to each other ico-chemical and functional properties of apple tea but distinctly different from the characteristics of wine.

(A) Rescaled Distance Cluster Combine Rescaled Distance Cluster Combine C A S E 0 5 10 15 20 25 (C) C A S E 0 5 10 15 20 25 Label Num Label Num

2 g Orthodox tea/100 ml apple juice 2 g Orthodox tea/100 ml apple juice (SC) 2 g Herbal tea/100 ml apple juice 2 g Herbal tea/100 ml apple juice (SC) 3 g Herbal tea/100 ml apple juice 3 g Herbal tea/100 ml apple juice (SC) 2 g CTC tea/100 ml apple juice 2 g CTC tea/100 ml apple juice (SC) 3 g Orthodox tea/100 ml apple juice 3 g Orthodox tea/100 ml apple juice (SC) 5 g Herbal tea/100 ml apple juice 4 g CTC tea/100 ml apple juice 5 g Herbal tea/100 ml apple juice (SC) 4 g Herbal tea/100 ml apple juice 4 g CTC tea/100 ml apple juice (SC) 3 g CTC tea/100 ml apple juice 4 g Herbal tea/100 ml apple juice (SC) 5 g Orthodox tea/100 ml apple juice 3 g CTC tea/100 ml apple juice (SC) 4 g Orthodox tea/100 ml apple juice 5 g Orthodox tea/100 ml apple juice (SC) 5 g CTC tea/100 ml apple juice 4 g Orthodox tea/100 ml apple juice (SC) 5 g CTC tea/100 ml apple juice (SC) (B) Rescaled Distance Cluster Combine 2 g Orthodox tea/100 ml apple juice (SC) C A S E 0 5 10 15 20 25 2 g Herbal tea/100 ml apple juice (SC) Label Num 5 g Orthodox tea/100 ml apple juice (SC) 5 g Herbal tea/100 ml apple juice (SC) 2 g CTC tea/100 ml apple juice 4 g CTC tea/100 ml apple juice (SC) 3 g CTC tea/100 ml apple juice 2 g CTC tea/100 ml apple juice (SC) 3 g Orthodox tea/100 ml apple juice 3 g CTC tea/100 ml apple juice (SC) 4 g Orthodox tea/100 ml apple juice 3 g Orthodox tea/100 ml apple juice (SC) 4 g Herbal tea/100 ml apple juice 4 g Orthodox tea/100 ml apple juice (SC) 3 g Herbal tea/100 ml apple juice 4 g Herbal tea/100 ml apple juice (SC) 5 g Orthodox tea/100 ml apple juice 3 g Herbal tea/100 ml apple juice (SC) 5 g Herbal tea/100 ml apple juice 5 g CTC tea/100 ml apple juice (SC) 4 g CTC tea/100 ml apple juice 2 g Orthodox tea/100 ml apple juice 2 g Herbal tea/100 ml apple juice SC = Fermentation with Saccharomyces cerevisiae var. ellipsoideus 5 g CTC tea/100 ml apple juice NF = Natural fermentation

Figure 3 - Dendrograms of different treatments of apple tea wine fermented with Saccharomyces cerevisiae var. ellipsoideus (A), by natural fermentation (B) and their comparison (C) using physico-chemical and functional properties analysed based on rescaled distance. CTC crush, tear and curl

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